Background: Safety is a concern that must be addressed prior to any clinical use of human immunodeficiency virus (HIV)-based lentiviral vectors in human patients. Unfortunately, efforts to examine the biosafety of the vectors in preclinical animal models are hampered due to the lack of animal models for HIV infection. We have developed new lentiviral vectors based on the recently characterised Jembrana Disease Virus (JDV), which infects a specific species of cattle naturally in Bali, Indonesia.
Methods: Sequences from the JDV genome were amplified by splicing overlap extension polymerase chain reaction (PCR) for the construction of transfer vectors as well as a packaging construct. Co-transfection of these two plasmids into 293T cells with a third encoding a G glycoprotein of vesicular stomatitis virus produced pseudotyped, disabled, replication defective JDV vector particles. Viral titre was obtained by transducing the cells with the supernatant harvested from transfectants and determining the number of cells expressing the transgene. PCR and Southern blotting were used to detect the presence of potential replication-competent viruses as well as transgene integration.
Results: Bicistronic JDV vectors encoding the green fluorescent protein (GFP) and the neomycin phosphotransferase were harvested with a titre range of 0.4-1.2 x 10(6) colony forming units/ml from vector-producing cells and were further concentrated by ultracentrifugation to the high titre of approximately 10(7) CFU/ml. Vectors encoding GFP were shown to transduce and integrate efficiently into the chromosomes of a range of primary and transformed cells of different origins in different differentiation status, including growth-arrested cells, with an efficiency of 25-75%. Exhaustive testing with a marker gene transfer assay in combination with a reverse transcriptase assay and PCR amplification of samples of serially passaged, transduced cells showed that no detectable amount of replication competent lentivirus (RCL) was produced.
Conclusions: We showed the feasibility of the development of gene transfer vectors based on a non-primate bovine lentivirus, which will provide the opportunity for examination of the efficacy and biosafety of lentiviral vector-mediated gene transfer in vivo in animal models. JDV-based vectors may be applicable and more readily acceptable than those from HIV for human gene therapy.